The aim of this grant proposal is to provide ameliorate the effects of mustard using high dose vitamin D3 as a countermeasure, specifically to combat macrophage hyper-activation in the skin. The study has accomplished it goals in establishing 2 mouse dermal models of sulfur mustard and nitrogen mustard skin injury. They study has also completed the first-in-human clinical trial to demonstrate the efficacy of vitamin D3 to promote skin repair from inflammation from UV exposure. In the UV dose finding trial, it was learned that 200,000 I.U. of vitamin D3 is required for promoting skin repair. In the final year of this proposal, healthy subjects were exposed to small patch testing of topical mechlorethamine (medicinal nitrogen mustard) with the goal of determining whether vitamin D3 is effective against such exposure. The trial has been completed and closed to enrollment. Skin and serum have been procured and are poised for analysis to elucidate biomarkers and transcription factors in NM exposed skin using RNA-seq and ChIP-seq. Since human studies are both complicated and delicate with generation of precious tissue samples, the new discoveries will allow us to better define meaningful biomarkers and endpoints for future studies. Nitrogen mustard mediated tissue injury of the skin results in rapid epidermal disruption and vascular leakage resulting edema and painful inflammation. Following the initial injury is an influx of activated inflammatory monocytes and macrophages. Release of macrophage-mediated soluble signaling molecules promotes a pro-inflammatory state which amplifies other cytotoxic activities including release of reactive nitrogen species iNOS, reactive oxygen species, and induction of proteolytic enzymes. Furthermore, these activated iNOS-producing macrophages are critically involved in mediating and exacerbating systemic toxicities. We have demonstrated in preclinical studies that vitamin D3 can inhibit the production of pro- inflammatory factors including; TNF?, and iNOS in the skin and in activated macrophages. This is a novel and highly translational approach to develop potential countermeasure to address key morbidity and mortality factors from mustard exposure. Vitamin D3 is widely available and inexpensive and, if shown to be beneficial, could have a broad public health impact in the event of chemical weapon exposure.